Calcium, mitochondria and apoptosis studied by fluorescence measurements.
Among the many unsolved problems of calcium signalling, the role of calcium elevations in apoptotic and necrotic cell death has been a focus of research in recent years. Evidence has been presented that calcium oscillations can effectively trigger apoptosis under certain conditions and that dysregulation of calcium signalling is a common cause of cell death. These effects are regularly mediated through calcium signal propagation to the mitochondria and the ensuing mitochondrial membrane permeabilization and release of pro-apoptotic factors from mitochondria to the cytoplasm. The progress in this area depended on the development of (1) fluorescent/luminescent probes, including fluorescent proteins that can be genetically targeted to different intracellular locations and (2) the digital imaging technology, fluorescence-activated cell sorting and fluorescent high throughput approaches, which allowed dynamic measurements of both [Ca2+] in the intracellular compartments of interest and the downstream processes. Fluorescence single cell imaging has been the only possible approach to resolve the cell-to-cell heterogeneity and the complex subcellular spatiotemporal organization of the cytoplasmic and mitochondrial calcium signals and downstream events. We outline here fluorometric and fluorescence imaging protocols that we set up for the study of calcium in the context of apoptosis.
Roy
Soumya Sinha
SS
Department of Pathology, Anatomy and Cell Biology, Thomas Jefferson University, 1020 Locust Street, Suite 253 JAH, Philadelphia, PA 19107, USA.
Hajnóczky
György
G
D000818:Animals; D017209:Apoptosis / Q000502:physiology; D002118:Calcium / Q000502:physiology*; D020013:Calcium Signaling / Q000502:physiology*; D020169:Caspases / Q000378:metabolism; D016923:Cell Death; D002460:Cell Line; D004072:Digitonin / Q000494:pharmacology; D005456:Fluorescent Dyes; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D008856:Microscopy, Fluorescence; D008928:Mitochondria / Q000502:physiology; D033681:Mitochondrial Membrane Transport Proteins / Q000502:physiology; D051336:Mitochondrial Membranes; D000083162:Mitochondrial Permeability Transition Pore; D009336:Necrosis / Q000378:metabolism; D010539:Permeability / Q000187:drug effects; D051381:Rats
D005456:Fluorescent Dyes; D033681:Mitochondrial Membrane Transport Proteins; D000083162:Mitochondrial Permeability Transition Pore; D049452:Green Fluorescent Proteins; D020169:Caspases; D004072:Digitonin; D002118:Calcium